AMPK phosphorylation proceeds through hierarchical proteoform cascades revealed by integrated mass spectrometry

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Protein kinases integrate cellular signals through complex phosphorylation cascades, yet resolving how chemical perturbations trigger and modulate these cascades in therapeutic targets remains a major challenge. Here, we dissect adenosine 5'-monophosphate-activated protein kinase (AMPK) proteoforms during activation through controlled biochemical reactions with a hybrid mass spectrometry (MS) approach integrating bottom-up MS for site-specific kinetics with top-down proteoform characterization. We reveal that AMPK phosphorylation proceeds through hierarchical cascades rather than binary switching, with dual entry points: canonical calcium- and calmodulin-dependent protein kinase kinase 2 (CaMKK2)-mediated phosphorylation or allosteric activator PF-739 both triggering extensive autophosphorylation with α1-Ser496 (S496) showing the highest kinetic priority. Proteoform-resolved analysis uncovers channeled β1-Ser24/25 (S24/25) + Ser108 (S108) cophosphorylation linking subcellular localization with allosteric responsiveness. Site-directed mutagenesis demonstrates CaMKK2 targets only α1-Thr183 (T183), with all other modifications arising through autophosphorylation. Phosphatase competition reveals asymmetric control where PP1A selectively removes activation-loop phosphorylation while autophosphorylation sites remain protected, establishing persistent regulatory states. Resolving AMPK's temporal kinetics and proteoform architecture during activation enables a proteoform-centric understanding on kinase regulation.

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